Red rot resistance in interspecific protoplast fusion product progeny of Porphyra yezoensis and P. tenuipedalis (Bangiales, Rhodophyta)

Nine primary regenerants were recovered by interspecific protoplast fusion of Porphyra yezoensis Ueda T‐14 (Py) (cultivated Porphyra) and Porphyra tenuipedalis Miura (Pt). This combination is difficult to achieve with conventional sexual hybridization, yet is important in that non‐cultivated P. tenuipedalis is partially resistant (PR) to red rot disease, caused by the microbial pathogen, Pythium porphyrae Takahashi et Sasaki. Out of the nine primary regenerants, two strains (Py‐Pt‐4 and Py‐Pt‐7) were like the parent, P. tenuipedalis, while the rest were like the other cultivated parent P. yezoensis T‐14 in their life cycle. Red rot resistance was assessed in parents and interspecific fusion product progeny (FPP) by exposing the foliose thalli to equivalent infection and measuring two parameters of the host‐pathogen interactions: supported fungal biomass and amount of disease produced. Intermediate resistance between P. yezoensis T‐14 (1.00) and P. tenuipedalis (0.13) was observed in two of the Py‐type FPP, Py‐Pt‐2F₂ (0.25) and Py‐Pt‐5F₂ (0.23). Stable inheritance of resistance was observed through two subsequent generations. The morphologic and reproductive characteristics of the regenerated foliose thalli, and nature of host‐pathogen interactions were used to further verify the hybrid origin of the FPP. Host‐pathogen interactions were followed using epi‐fluorescence and scanning electron microscopy (SEM). The zoospores encysted at higher rates on the susceptible cultivated parent (P. yezoensis T‐14) germinated immediately and the short germ tubes formed appres‐soria and penetrated the algal cells near the site of encystment. While on the PR parental (P. tenuipedalis) and partially resistant FPP (PRFPP) progeny (Py‐Pt‐2F₂ and Py‐Pt‐5F₂) the low rate of zoospore encystment was followed by cyst germination, but only a few of the germ tubes formed appressoria and penetrated the thallus surface. Long germ tubes (with no appressoria) were seen growing on the thallus surface without host penetration. The minimal rate of encystment concomitant with low rate of appressorium formation on the PR parent and PRFPP was observed as the major factor responsible for the partial resistance in these thalli.     

Isolation of Porphyran-Degrading Marine Microorganisms from the Surface of Red Alga,Porphyra yezoensis

Marine microorganisms degrading porphyran (POR) were found on the surface of thalli of Porphyra yezoensis. Fifteen crude microorganism groups softened and liquefied the surface of agar-rich plate medium. Among these, 11 microorganism groups degraded porphyran that consisted of sulfated polysaccharide in Porphyra yezoensis. Following isolation, 7 POR-degradable microorganisms were isolated from the 11 POR-degradable microorganism groups.     

Serological detection of red rot disease initiation stages of microbial pathogen, Pythium porphyrae (Oomycota) on Porphyra yezoensis

Pythium porphyrae (Oomycota), a pathogen causing red rot diseasein Porphyra spp., can at present only be detected when colonizationof the host thallus has already occurred and so it is often too late to takeappropriate disease control measures. The paper presents an account of an effective methdology for early detection of the disease. Since Py.porphyrae zoospores are the primary means of pathogen dispersal,polyclonal antibodies (Pabs) were raised against the surface components ofzoospores and encysted zoospores. Using these Pabs the disease initiationstages of the Pythium porphyrae were detected on the surface of Porphyra thalli by immunofluorescence assay. The specificity of theseantibodies and the efficacy of immunofluorescence assay in the detectionof red rot disease are discussed.     

Taxonomy, molecular phylogeny, and ultrastructural morphology of Olpidiopsis porphyrae sp. nov. (Oomycetes, straminipiles), a unicellular obligate endoparasite of Bangia and Porphyra spp. (Bangiales, Rhodophyta)

Olpidiopsis porphyrae sp. nov., a marine oomycete endoparasite that infects the commercially cultivated red alga Porphyra yezoensis, is described and its phylogenetic position based on molecular data and ultrastructural morphology is discussed. O. porphyrae infects the host Porphyra by means of encysted zoospores. Spherical-shaped holocarpic thalli develop within the cytoplasm of its algal host, which produce monoplanetic, subapically biflagellate zoospores. The characteristic features of this isolate are the ellipsoidal, unicellular thallus and simple holocarpic zoosporangial development, which show morphological similarity with the genus Olpidiopsis. Laboratory infection experiments with a wide range of green, brown, and red algae revealed that O. porphyrae infects several stages of the bangialean red algae (the genera Bangia and Porphyra). Molecular phylogenetic analyses inferred from both SSU rRNA and cox2 genes showed O. porphyrae branched before the main saprolegnian and peronosporalean lineages within the monophyletic oomycete clade, indicating its phylogenetic separation from them. A single or double K-body-like organelle, which contains tubular inclusions, is found located to one side of the zoospore nucleus and shows similarities to homologous organelles previously described in O. saprolegniae. The ultrastructural morphology of O. porphyrae with zoospore initials containing K-bodies and tubular mitochondrial cristae is characteristic of oomycetes. Group I intron-like multiple insertions were found in the SSU rRNA gene of O. porphyrae. This is the first report of SSU group I introns in the class Oomycetes.     

Forecasting infections of the red rot disease on Porphyra yezoensis Ueda (Rhodophyta) cultivation farms

Pythium porphyrae is a fungal pathogen responsible for red rot disease of the seaweed Porphyra (Rhodophyta). Infection forecasts of Porphyra by P. porphyrae were estimated from the epidemiological observations of Porphyra thalli and numbers of zoospore of P. porphyrae in laboratory and cultivation areas. Four features of forecasting infections were determined by relating zoospore concentrations to the incidence of thallus infection; infection (in more than 1000 zoospores L⁻¹), microscopic infection [less than 2 mm in diameter of lesion (in from 2000 to 3000 zoospores L⁻¹)], macroscopic infection [more than 2 mm in diameter of lesion (in from 3000 to 4000 zoospores L⁻¹), and thallus disintegration (in more than 4000 zoospores L⁻¹). High zoospore concentrations led to more infection. The tendency that zoospore concentration of P. porphyrae increased with the rate of infection of Porphyra thalli was generally observed in forecasting infections in both the laboratory and in cultivation areas. Based on the Porphyra cultivation areas, the accuracy and consistency of forecasting infections suggest that this method could be employed to manage and control red rot disease.     

Rapid detection of Pythium porphyrae in commercial samples of dried Porphyra yezoensis sheets by polymerase chain reaction

The fungal parasite Pythium porphyrae is the causative organism of red rot disease in Porphyra cultivation farms. The detection of P. porphyrae from dried Porphyra yezoensis sheets was achieved using the species-specific primers PP-1 (5′-TGTGTTCTGTGCT-CCTCTCG-3′) and PP-2 (5′-CCCAAATTGGTGTTGCCTCC-3′) with the polymerase chain reaction (PCR). The DNA sequence (707 bp) of PCR product was found to be identical to that amplified from ITS rDNA extracted from a type species of P. porphyrae (IFO 30800, The Institute of Fermentation, Osaka, Japan). Quantities of the product amplified varied with the time when samples were harvested after the occurrence of red rot disease in Porphyra farms. This simple, rapid, and inexpensive method should have great applications in furthering quality control and determination of quality ranking in the Porphyra processing industry.     

VARIATIONS IN THE CELL WALLS AND PHOTOSYNTHETIC PROPERTIES OF PORPHYRA YEZOENSIS (BANGIALES,RHODOPHYTA) DURING ARCHEOSPORE FORMATION1

The formation of archeospores is characteristic of Porphyra yezoensis Ueda and is important for Porphyra aquaculture. Recently, it has been regarded as a valuable seed source for propagation of thalli in mariculture. Cell wall composition changes are associated with archeospore formation in P. yezoensis. Here, we report changes of cell walls of P. yezoensis during archeospore formation. The surfaces of vegetative cells that were originally smooth became rougher and more protuberant as archeosporangia were formed. Ultimately, the cell walls of archeosporangia ruptured, and archeospores were released from the torn cell walls that were left at distal margins of thalli. With changes in cell walls, both effective quantum yield and maximal quantum yield of the same regions in thalli gradually increased during the transformation of vegetative cells to archeospores, suggesting that the photosynthetic properties of the same regions in thalli gradually increased. Meanwhile, photosynthetic parameters for different sectors of thalli were determined, which included the proximal vegetative cells, archeosporangia, and newly released archeospores. The changes in photosynthetic properties of different sectors of thalli were in accordance with that of the same regions in thalli at different stages. In addition, the photosynthetic responses of archeosporangia to light showed higher saturating irradiance levels than those of vegetative cells. All these results suggest that archeosporangial cell walls were not degraded prior to release but were ruptured via bulging of the archeospore within the sporangium, and ultimately, archeospores were discharged. The accumulation of carbohydrates during archeospore formation in P. yezoensis might be required for the release of archeospores.     

GENETIC DIVERSITY AND INTROGRESSION IN TWO CULTIVATED SPECIES (PORPHYRA YEZOENSIS AND PORPHYRA TENERA) AND CLOSELY RELATED WILD SPECIES OF PORPHYRA (BANGIALES,RHODOPHYTA)1

We investigated the genetic variations of the samples that were tentatively identified as two cultivated Porphyra species (Porphyra yezoensis Ueda and Porphyra tenera Kjellm.) from various natural populations in Japan using molecular analyses of plastid and nuclear DNA. From PCR‐RFLP analyses using nuclear internal transcribed spacer (ITS) rDNA and plastid RUBISCO spacer regions and phylogenetic analyses using plastid rbcL and nuclear ITS‐1 rDNA sequences, our samples from natural populations of P. yezoensis and P. tenera showed remarkably higher genetic variations than found in strains that are currently used for cultivation. In addition, it is inferred that our samples contain four wild Porphyra species, and that three of the four species, containing Porphyra kinositae, are closely related to cultivated Porphyra species. Furthermore, our PCR‐RFLP and molecular phylogenetic analyses using both the nuclear and plastid DNA demonstrated the occurrence of plastid introgression from P. yezoensis to P. tenera and suggested the possibility of plastid introgression from cultivated P. yezoensis to wild P. yezoensis. These results imply the importance of collecting and establishing more strains of cultivated Porphyra species and related wild species from natural populations as genetic resources for further improvement of cultivated Porphyra strains.     

CRYOPRESERVATION OF THE CONCHOCELIS OF PORPHYRA (RHODOPHYTA) BY APPLYING A SIMPLE PREFREEZING SYSTEM1

The conchocelis cells of four strains of Porphyra yezoensis Udea and four other Porphyra species were cryopreserved in liquid nitrogen (LN) using a programmable freezer or a simple prefreezing system, which consisted of a styrofoam box and a deep-freezer at ?40° C. The cells differed in their freezing tolerance but survived maximally when prefrozen to ?40° C in a cryoprotective solution composed of 10% dimethylsulfoxide and 0.5 M sorbitol in 50% seawater. The cryopreservation was successfully performed by applying the simple prefreezing system as well as by a programmable freezer. Conchocelis cells thawed from the LN temperature formed colonies and retained the ability to form conchospores that grew into gametophytic thalli. This technique using a simple prefreezing system will accelerate the spread of Porphyra cryopreservation.     

Detection and quantitative analysis of zoospores of Pythium porphyrae, causative organism of red rot disease in Porphyra, by competitive PCR

The detection and quantitative analysis of Pythium porphyraezoospores was performed by PCR using PP-1 and PP-2 primers specific tothe internal transcribed spacer region of P. porphyrae. To estimatethe amount of fungal zoospores of P. porphyrae, an internal standardplasmid (pPPISC) containing a modified DNA fragment was constructed. Both ends of this fragment were complementary to the PCR primers. Amplification using primers PP-1 and PP-2 produced DNA fragments ofapproximately 700 and 400 bp from the target DNA of P. porphyraezoospores and from the pPPISC, respectively. To perform quantitativePCR, known quantities of pPPISC were added to reaction mixturescontaining the experimental DNAs extracted from zoospores. After aco-amplification reaction, the two different sized PCR products wereseparated by agarose gel electrophoresis and visualized by ethidium bromidestaining. The number of zoospores was estimated by comparing thefluorescence intensities of the PCR products using a charge-coupled deviceimage analyzer. The results show that competitive PCR using P.porphyrae specific primers and competitor pPPISC are useful tools for thequantitative analysis of P. porphyrae zoospores in seawater from Porphyra cultivation farms.     

TWO SPECIFIC CAUSES OF CELL MORTALITY IN FREEZE‐THAW CYCLE OF YOUNG THALLI OF PORPHYRA YEZOENSIS (BANGIALES,RHODOPHYTA)1

Porphyra yezoensis Ueda is an important marine aquaculture crop with single‐layered gametophytic thalli. In this work, the influences of thallus dehydration level, cold‐preservation (freezing) time, and thawing temperature on the photosynthetic recovery of young P. yezoensis thalli were investigated employing an imaging pulse‐amplitude‐modulation (PAM) fluorometer. The results showed that after 40 d of frozen storage when performing thallus thawing under 10°C, the water content of the thalli showed obvious effects on the photosynthetic recovery of the frozen thalli. The thalli with absolute water content (AWC) of 10%–40% manifested obvious superiority compared to the thalli with other AWCs, while the thalli thawed at 20°C showed very high survival rate (93.10%) and no obvious correlation between thallus AWCs and thallus viabilities. These results indicated that inappropriate thallus water content contributed to the cell damage during the freeze‐thaw cycle and that proper thawing temperature is very crucial. Therefore, AWC between 10% and 40% is the suitable thallus water content range for frozen storage, and the thawing process should be as short as possible. However, it is also shown that for short‐term cold storage the Porphyra thallus water content also showed no obvious effect on the photosynthetic recovery of the thalli, and the survival rate was extremely high (100%). These results indicated that freezing time is also a paramount contributor of the cell damage during the freeze‐thaw cycle. Therefore, the frozen nets should be used as soon as time permits.     

BIOCHEMICAL PHENOTYPES CORRESPONDING TO MOLECULAR PHYLOGENY OF THE RED ALGAE PLOCAMIUM (PLOCAMIALES,RHODOPHYTA): IMPLICATIONS OF INCONGRUENCE WITH THE CONVENTIONAL TAXONOMY1

Among five species of the genus Plocamium Lamouroux distributed around Japan, P. cartilagineum (Linnaeus) Dixon, P. recurvatum Okamura and P. telfairiae (Hooker and Harvey) Harvey are often difficult to distinguish morphologically from each other. Our previous study demonstrated that P. recurvatum and P. telfairiae were divided into two groups, A and C, based on RUBISCO spacer sequence and that the specimens belonging to group C had acidic cell saps. In this study, we inferred evolutionary relationships of these Plocamium species from internal transcribed spacer sequence of the ribosomal RNA genes and obtained a similar topology to the RUBISCO spacer tree. Color of the dried specimens in the acidic group C was darker red than that in the non‐acidic group A, although there was no difference in color in living thalli. The Br^? concentration in the cell sap of the acidic group C was 20 times higher than that of the non‐acidic group. We could not find any morphological differences to distinguish clearly between groups A and C despite exhaustive investigation of field‐collected and cultured thalli in both P. recurvatum and P. telfairiae. These results suggest that the color of dried specimens and the composition of intracellular inorganic ions are significant criteria for interpreting phylogenetic relationships in Japanese Plocamium spp.     

Activation of Murine Macrophages by Polysaccharide Fractions from Marine Algae (Porphyra yezoensis)

Two discrete immunomodulating fractions were obtained from marine algae (Porphyra yezoensis): one was the Porphyra water-soluble fraction (PWSF) which was extracted with hot water from the whole body of algae, and the other was the Porphyra acid-soluble fraction (PASF) which was extracted with acid from the residue. The major constituent in both PWSF and PASF was a polysaccharide, the total sugar concentration in PWSF (56.4%) being lower than that in PASF (82.2%). The high contents of 3,6-anhydrogalactose and sulfate indicated the porphyran structure in PWSF and PASF. The results of an in vitro culture assay with proteose peptone-induced macrophages from mice revealed that PWSF and PASF both enhanced glucose consumption, as well as the production of nitrite and tumor necrosis factor (TNF), but that these were increased more by PWSF than by PASF. PWSF augmented IL-1 secretion from these macrophages, while PASF did not. On the other hand, the carbon clearance activity of phagocytes from mice injected intraperitoneally with PASF was higher than that from PWSF-injected mice. The injection of PASF into mice also enhanced the carbon clearance activity in a dose-dependent manner. These results suggest that the two individual fractions possessed the ability to activate macrophages in vitro and in vivo in different ways.     

Genetic variation in 14 Porphyra lines using restriction site amplified polymorphism (RSAP)

Restriction site amplified polymorphism (RSAP) is a molecular marker technique which just requires a simple polymerase chain reaction to amplify fragments around restriction sites. The RSAP analytic system was set up and applied to Porphyra genetic variation analysis in this study for the first time. Fourteen Porphyra lines were screened by the RSAP analytic system with 30 primer combinations, 12 of which produced stable and reproducible amplification patterns in three repeated experiments. The 12 primer combinations produced 408 amplified fragments, 402 of which (98.53%) were polymorphic, with an average of 33.5 polymorphic fragments for each primer combination, ranging in size from 50 to 500 bp. The 408 fragments were scored one by one and then used to develop a dendrogram of the 14 Porphyra lines with unweighted pair-group method arithmetic average. The genetic distance among these Porphyra lines ranged from 0.10 to 0.50. These Porphyra lines were divided into two major groups at the 0.71 similarity level: one group contained only Porphyra haitanensis lines and the other group contained Porphyra yezoensis lines. In addition, some specific RSAP markers were acquired from each Porphyra line apart from P. yezoensis Yqd-2-1, and five of them were sequenced. One of the specific markers, R1/R3-8₁₁₉ from P. yezoensis Y-9101, was successfully converted into sequence characterized amplification region marker. The result suggested that TRAP was a simple, stable, polymorphic, and reproducible molecular marker technique for the classification and resource protection of Porphyra lines.     

Nuclear division of the vegetative cells, conchosporangial cells and conchospores of Porphyra yezoensis (Bangiales, Rhodophyta)

To confirm the position and timing of meiosis in Porphyra yezoensis Ueda, the nuclear division of vegetative cells, conchosporangial cells and conchospores was observed. An improved staining method using modified carbol fuchsin was introduced to stain the chromosomes of Porphyra. Pit‐connections between conchosporangial cells also stained well with this method. Leptotene, zygotene, pachytene, diplotene, diakinesis, metaphase, anaphase and telophase were observed in the conchosporangial cells. During the germination of conchospores, no characteristics of meiosis I were found. No difference between the nuclear division of vegetative cells and that of conchospores was observed, and 2–3 days were needed for the first cell division both in vegetative cells and conchospores. Therefore, the cell division that occurs during conchospore germination is not meiosis I. Our results indicate that the prophase of meiosis I begins during the formation of conchosporangial branches, and metaphase I, anaphase I and telophase I take place during the maturation of conchosporangial branches. Then the three‐bivalent nucleate sporangia complete cell division to form two individual conchospores, each with one three‐univalent nucleus. The conchospores released from the sporangia are at meiotic interphase. Meiosis II occurs at the first nuclear division during conchospore germination, which is a possible explanation for the observation of mosaic thalli in mutant germlings of P. yezoensis. The mosaic thalli might also arise from gene conversion/post meiotic segregation events, comparable to those in Sordaria fimicola (Roberge ex Desm.) Ces. & De Not. and Neurospora crassa Shear & B.O. Dodge.     

New species of unicellular obligate parasite, <Emphasis Type="Italic">Olpidiopsis pyropiae</Emphasis> sp. nov., that plagues <Emphasis Type="Italic">Pyropia</Emphasis> sea farms in Korea

Pyropia (Porphyra) sea farms are plagued with many diseases, similar to a land crop field. Olpidiopsis disease has been one of the most serious diseases causing multimillion dollars of economic loss every year. From 3 years of epidemiological studies in Pyropia sea farms, we found that the pathogen of Olpidiopsis disease in Korea is different from the oomycete, Olpidiopsis porphyrae, which is known to infect the commercially cultivated Pyropia yezoensis in Japan. The Korean species showed a clearly different small subunit (SSU) 18S rRNA sequence (90.4 % identity) and lacked four intron-like insertions, which are present in O. porphyrae. We therefore established a new species named Olpidiopsis pyropiae. The infection process and asexual life history of O. pyropiae were similar to O. porphyrae. Infection started when zoospores attached to the surface of Pyropia blade, lost flagella, and produced thin germ tubes that penetrated the cell walls of the host. Spherical multinucleate thalli developed within the cytoplasm of its algal host, which grew into mature sporangia within the next 2 days. The shape and size of sporangium was similar to that of the Japanese species, except for longer discharge tubes in O. pyropiae. Transmission electron microscopy revealed the presence of K-body-like organelles with tubular inclusions located close to the nucleus, which is one of the key characters of the genus. However, phylogenetic analysis based on SSU ribosomal RNA (rRNA) gene data showed a loose affinity of the Korean species with the other marine Olpidiopsis spp.     

Preliminary comparison of atmospheric CO2 enhancement to photosynthesis of Pyropia yezoensis (Bangiales,Rhodophyta) leafy thalli and filamentous thalli

Intertidal macroalgae are submerged in seawater at high tide and exposed to air at low tide. When they are exposed to the air, CO₂ is the main inorganic carbon source. In this study, the photosynthetic performances of PSI and PSII were measured in different generations of Pyropia yezoensis (leafy thalli and filamentous thalli) that had been exposed to air containing different CO₂ concentrations. Changes in photosynthesis during dehydration and salt treatment under the different CO₂ concentrations were also analyzed. The results showed that in leafy thalli, the effective photochemical quantum yield of PSII (YII) was enhanced as CO₂ increased, which suggested that CO₂ assimilation was enhanced and that they can utilize CO₂ in the air directly, even when they are subjected to moderate stress. These findings could explain why, in P. yezoensis aquaculture, moderate exposure to air does not lead to a decrease in crop yield. However, in filamentous thalli, there were no significant differences in YII at the CO₂ concentrations tested. The expression of genes involved in the Calvin cycle in leafy thalli was higher than that in filamentous thalli. CO₂ uptake and biomass of P. yezoensis leafy thalli is larger than filamentous thalli, which may be due to its different carbon utilization mechanism and the adaptation of intertidal environment in the evolutionary process.     

Conchospore production and seasonal occurrence of some Porphyra species (Bangiales,Rhodophyta) in Washington State

The leafy thalli of species of the marine red algal genus Porphyra grow rapidly but persist for a relatively short time on rocky intertidal or subtidal substrata or as epiphytes on other marine plants. In most species, the large, short-lived leafy thalli alternate with small, presumably perennial, filamentous conchocelis plants. Depending on the species of northeastern Pacific Porphyra, photoperiod and temperature are important regulators of conchospore formation and release. Data from laboratory studies of conchospore formation and release in five Washington species of Porphyra (P. abottae, P. nereocystis, P. perforata, P. pseudolanceolata and P. torta) indicate that conchospores are most likely to be released at a time that precedes the appearance of the leafy thalli in the field.     

MORPHOLOGICAL AND MOLECULAR ANALYSIS OF THE ENDANGERED SPECIES PORPHYRA TENERA (BANGIALES,RHODOPHYTA)1

Porphyra tenera Kjellman, widely cultivated in nori farms before the development of artificial seeding, is currently listed as an endangered species in Japan. To confirm whether a wild‐collected gametophytic blade was P. tenera or the closely related species P. yezoensis Ueda, morphological observations and molecular analyses were made on the pure line HGT‐1 isolated from a wild blade. This pure line was identified as P. tenera based on detailed morphological features. Sequences of the nuclear internal transcribed spacer region 1 and the plastid RUBISCO spacer revealed that P. tenera HGT‐1 was clearly different from P. yezoensis f. narawaensis Miura, the main species cultivated in Japan. PCR‐RFLP analysis of the internal transcribed spacer region was found to be a convenient method for rapid discrimination between P. tenera and cultivated P. yezoensis. The restriction patterns generated by the endonucleases Dra I and Hae III were useful for differentiating between both gametophytic and conchocelis stages of P. tenera HGT‐1 and P. yezoensis f. narawaensis strains. Thus, PCR‐RFLP analysis will serve as a valuable tool for rapid species identification of cultivated Porphyra strains, culture collections of Porphyra strains for breeding material and conservation of biodiversity, and, as codominant cleaved amplified polymorphic sequence markers for interspecific hybridization products between P. tenera and P. yezoensis f. narawaensis. Under the same culture conditions, rate of blade length increase and the blade length‐to‐width ratio were lower in P. tenera HGT‐1 than in P. yezoensis f. narawaensis HG‐4. The HGT‐1 became mature more rapidly than HG‐4 and had thinner blades.     

A CELLULOSE SYNTHASE (CESA) GENE FROM THE RED ALGA PORPHYRA YEZOENSIS (RHODOPHYTA)1

The cell walls of Porphyra species, like those of land plants, contain cellulose microfibrils that are synthesized by clusters of cellulose synthase enzymes (“terminal complexes”), which move in the plasma membrane. However, the morphologies of the Porphyra terminal complexes and the cellulose microfibrils they produce differ from those of land plants. To characterize the genetic basis for these differences, we have identified, cloned, and sequenced a cellulose synthase (CESA) gene from Porphyra yezoensis Ueda strain TU‐1. A partial cDNA sequence was identified in the P. yezoensis expressed sequence tag (EST) index using a land plant CESA sequence as a query. High‐efficiency thermal asymmetric interlaced PCR was used to amplify sequences upstream of the cDNA sequence from P. yezoensis genomic DNA. Using the resulting genomic sequences as queries, we identified additional EST sequences and a full‐length cDNA clone, which we named PyCESA1. The conceptual translation of PyCESA1 includes the four catalytic domains and the N‐ and C‐terminal transmembrane domains that characterize CESA proteins. Genomic PCR demonstrated that PyCESA1 contains no introns. Southern blot analysis indicated that P. yezoensis has at least three genomic sequences with high similarity to the cloned gene; two of these are pseudogenes based on analysis of amplified genomic sequences. The P. yezoensis CESA peptide sequence is most similar to cellulose synthase sequences from the oomycete Phytophthora infestans and from cyanobacteria. Comparing the CESA genes of P. yezoensis and land plants may facilitate identification of sequences that control terminal complex and cellulose microfibril morphology.